Quantification of human polyomavirus JC virus load in urine and blood samples of healthy tribal populations of North-Eastern part of West Bengal, India

S Chattaraj1, NK Bera2, C Dutta3, S Bhattacharjee11 Department of Zoology, University of North Bengal, Laboratory of Cell and Molecular Biology, Darjeeling, West Bengal, India2 Department of Psychiatry, North Bengal Medical College and Hospital, Darjeeling, West Bengal, India3 Department of Nephrology, North Bengal Medical College and Hospital, Darjeeling, West Bengal, India

Date of Submission

28-Oct-2014

Date of Acceptance

06-May-2015

Date of Web Publication

16-Oct-2015

Correspondence Address:S BhattacharjeeDepartment of Zoology, University of North Bengal, Laboratory of Cell and Molecular Biology, Darjeeling, West Bengal India

Source of Support: Council of Scientific and Industrial Research,
New Delhi, India, Conflict of Interest: None

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DOI: 10.4103/0255-0857.167345

~ Abstract

Background: Human polyomavirus JC (JCV) is a widespread human virus with profound pathogenic potential. A study was undertaken to quantify JCV load in urine and peripheral blood samples of immunocompetent, apparently healthy tribal individuals of North-Eastern part of West Bengal, India for the first time. Materials and Methods: One hundred and thirteen samples of urine or blood were collected from different tribal groups of this region. For the quantitative estimation of the viral load in each sample, real-time polymerase chain reaction method using the SYBR Green dye was employed. Results: The viral load estimated was found in the range between 3.5 × 102 and 2.12 × 106 copies/ml of samples having a mean and median viral copy numbers of 8.67 × 105 and 9.19 × 105 copies/ml of sample respectively. Conclusion: The mean viral DNA load in urine samples of the studied immunocompetent population was found to be higher than that found in a study conducted in the USA, but lower than similar groups of Italy and healthy adult women in the USA. However when compared with median values of viral DNA loads in urine samples of immunocompetent human subjects of Kuwait, Portugal, and Switzerland the observed viral DNA load was found to be substantially higher.

JC virus (JCV) is a member of the Polyomaviridae family. Apart from few isolated human populations JCV appears to be widespread worldwide.[1],[2] Initial infection with the virus occurs in childhood, after which the virus may persist in the kidneys and lymphoid tissues.[3] It is associated with a fatal demyelinating central nervous system (CNS) infection called progressive multifocal leukoencephalopathy (PML) in immunocompromised hosts such as in patients with AIDS [4] and involves productive infection in both oligodendrocytes and astrocytes.

The genome of the virus is a covalently closed, circular double-stranded DNA molecule having a length of about 5000 base pair.[5] It comprises of early and late coding region controlled by a common non-coding control region (NCCR). The "early" region encodes for the viral tumour antigens (T/t-antigens) and is expressed prior to initiation of DNA replication and the "late" region encodes three viral capsid proteins (VP1, VP2 and VP3).

Conventional polymerase chain reaction (PCR) has been used to detect the presence of JCV in both immunocompromised, as well as immunocompetent individuals. With the development of real-time PCR technology quantitation of viral nucleic acid loads in body fluids has become accurate.[6] However, report of quantitation of JCV load in immunocompetent subjects is scanty.

This study was done on the apparently healthy tribal groups of the North-Eastern part of West Bengal, India to estimate the quantity of viral load in individuals infected with the virus. For the quantitation of the virus, real-time PCR method was employed and oligonucleotide primers specific for the VP1 region of the viral genome was used. In our earlier study, an overall incidence rate of 25% was observed in the Oraon, Munda and Rabha tribal groups of this region.[7] However, in the present study, the incidence rate of the virus was found to be a little lower due to the incorporation of Mech population with the previously studied tribal groups and was observed to be 18.58% in all of the tribal population taken together. The viral copy number value was observed to be in the range between 3.5 × 102 and 2.12 × 106 copies/ml of sample having a mean and median copy number value of 8.67 × 105 and 9.19 × 105 copies/ml of sample respectively.

~ Materials and Methods

Sample collection

The methodology was approved by our Institutional Human Ethical Committee. Urine and blood samples from 113 individuals of healthy tribal groups of the North-Eastern part of West Bengal State of India were collected with their prior informed consent. The tribal groups studied here included Oraon and Mundas of Darjeeling district, Rabha or Rava population of Cooch Behar district and Mech population of Jalpaiguri district. Samples were collected on a random basis and collection depended on the availability and willingness of the tribal subjects concerned. Three millilitre of urine specimen from all the 113 individuals were collected in Tarson 1.5 ml microfuge tubes and 1 ml of blood specimen from 53 individuals of Rava group were collected in 5 ml Tarson vials containing ethylenediaminetetraacetic acid and were brought to the laboratory in ice box and stored at −20°C refrigerator till DNA isolation.

DNA isolation from both the urine and blood samples was done using the High Pure Viral Nucleic Acid Kit (Roche Diagnostic GmbH, Germany) as per the manufacturer's instruction and the isolated DNA samples were stored at −20°C refrigerator until used for quantitation of the viral load. Detection of the JCV positive samples was done primarily by normal PCR using primers specific for the amplification of NCCR of the viral genome for qualitative detection and then the quantitative approach was employed using primers specific for VP1 region. The amplified products were analysed by agarose gel electrophoresis. The Chi-square test was employed using SPSS (version 20) [IBM Corporation, New York, USA] to compare between the JCV positivity status with that with the gender of the subjects studied. For the analyses, the P < 0.05 were considered significant.

Standard viral DNA

Plasmid pMITC-BSMKS carrying the full length of JC viral genome (JCV Mad-1 strain; 5130 base pairs), a gift from Richard J. Frisque, Department of Biochemistry, Microbiology, Molecular and Cell Biology, Pennsylvania State University, USA was used as the standard DNA for the quantitation of the viral load by real-time PCR.

Estimation of viral load by real-time polymerase chain reaction

Estimation of the viral DNA load in 13 out of the 21 positive DNA samples isolated from both urine and blood were done with the real-time PCR method. To perform this reaction, two primers namely JCVRVF (5ˈ-TCAATGGATGTTGCCTTTACTTT-3ˈ) and JCVRVR (5ˈ-ACGGGGTCCTTCCTTTCTC-3ˈ) specific for the VP1 region of the viral genome [8] were used, which produced an amplification product of about 109 bp lengths. The specificity of the primers for JCV VP1 was checked by nr-BLAST search. The reaction was carried out in Applied Biosystems Thermal Cycler, StepOne Plus ™. Each PCR reaction mixture consisted of 3 µl of both forward and reverse primers (2 pM), 10 µl of 2X SYBR Green Reagent (FastStart Universal SYBR Green Master [Rox], Roche, Germany) and 4 µl of DNA to a final volume of 20 µl. PCR protocol included the following: Initial heating at 95°C, 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 63°C for 30 s and extension at 72°C for 45 s. All the samples were run in duplicate. The cloned JCV Mad1 plasmid containing the whole viral genome in a 10 fold dilution series served as the standard DNA for the estimation. It was used to generate the quantification standard curve. The copy number of the plasmid DNA (pMITC-BSMKS; 8091 base pairs) was adjusted to 8.08 × 109, 8.08 × 108, 8.08 × 107, 8.08 × 106, 8.08 × 105, 8.08 × 104 copies/ml (in six tubes) following real-time PCR software, based on the concentration and the plasmid size. The sensitivity or lower detection limit of the assay was found to be 8.08 × 104 copies/ml of the plasmid. Standard curve plot is the quantity of standard viral DNA against cycle threshold (CT), where CT is defined as the first cycle in which amplification signal is detected over the mean base line. Mean base line is calculated from ΔRn values of 40 cycles.

~ Results

The study was carried out on apparently healthy tribal groups of North-Eastern part of West Bengal, India that included Oraon and Munda tribes from Kiran Chandra Tea Garden, Naxalbari, Darjeeling district, Rabha tribes from Poro busty, Tufangunj, Cooch Behar district and Mech tribes from Mendabari, Kalchini, Jalpaiguri district. An overall incidence rate of 18.58% was observed in the population of this region [Table 1]. Oraon and Munda groups showed a higher incidence of JCV, that is, 29.16% compared to the other groups studied. The possible association of virus prevalence with the gender of the subjects were statistically analysed. The frequency of JCV detection seems to differ between the genders and it has been found to be greater in males compared to the females in the tribes of this region with a P value of 0.003. In case of mech, due to the low positive detection of JCV (5.55%) among the random sample, the association between virus status and sex were not significant enough (P = 0.871). The viral load was estimated in the positive samples by real-time PCR method using primers specific for VP1 region of the JCV genome. The details regarding the copy number and CT value range of the JCV positive individuals are represented in [Table 2], which also includes mean and median DNA load of the virus in both urine and blood specimen of the population studied. A wide range of viral load was found in the samples ranging from 3.52 × 102 to 2.12 × 106 copies/ml of sample. The mean and median copy number values were found to be 8.67 × 105 copies/ml of sample and 9.19 × 105 copies/ml of sample respectively.

Table 1: Summary of the results showing incidences of the JCV in the studied tribal groups

JC virus is widely spread in the human population [9] and is the causative agent of PML in individuals with suppressed immune system. Because of its pathogenic role in humans, a study regarding the prevalence of the virus among the normal, apparently healthy tribal groups of North-Eastern part of West Bengal, India has already been done.[7] This study aimed at quantitation of the viral load in the immunocompetent healthy tribal populations of North-Eastern part of West Bengal, India.

The North Bengal region (North-Eastern parts of West Bengal, India) is inhabited for long by several tribal groups like Santhal, Oraon, Munda, Rabha, Chik-Baraik, Toto, Koch, Mech, Bhutia and others who belong to different linguistic groups, Austro-Asiatic, Dravidian and Tibeto-Chinese.[10] Oraons are the second largest and the Mundas are the third largest tribal population after the Santhals, constituting about 14% and 7.8% respectively of the total tribal population of West Bengal State of India.[11]

Traditionally, conventional PCR have been used for the detection of the JCV DNA in all types of samples. However, estimation of viral load by conventional PCR is at best semi-quantitative. Real-time PCR method has emerged as a new tool for both detection and quantification of the viral DNA isolated from different types of specimen.[6] In this study, we have used SYBR Green dye for quantitation and primers specific for the amplification of a 109 bp fragment in VP1 region of the JCV genome.

Prevalence of JCV has been recorded in the tribal population worldwide showing variations in the incidence range of the virus in different groups such as Native Americans,[12] tribals of Africa [13] and Myanmar tribals.[14] Previously we have documented a relatively higher incidence (~25%) of JCV DNA in the urine and peripheral blood leucocytes of Oraon, Munda and Rabha tribal groups of the North-Eastern part of West Bengal, India.[7] However, the overall incidence rate of the virus was now found to be 18.58% with the inclusion of Mech population, which had lower incidences of JCV reactivation (5.55%). The association between the JCV prevalence and the gender of the subjects was studied using Chi-square test. The frequency of the virus prevalence vary between the genders and is higher in case of males (P=0.003), which is in accordance with the previous studies done on Native Americans [12] and tribals of Africa.[13] The quantitation of viral DNA load present in the body fluids viz., urine and blood of the tribal populations of this region is being reported now for the first time in this study.

We have recorded a wide range of viral copy numbers in the urine and blood samples from the population of this region, which ranged from 3.52 × 102 to 2.12 × 106 copies/ml of sample. Individuals infected with the virus were found to have a high amount of viral DNA load present in their urine and blood specimens. The mean (8.67 × 105) and the median (9.19 × 105) viral copy number recorded in the present study was higher than observed in the some of the studies done earlier on healthy immunocompetent subjects. The mean (8.67 × 105 copies/ml of sample) viral load in the samples was comparatively higher than normal healthy subjects of USA having a value of only 3 × 103 copies/ml of sample.[15] The median value, 9.19 × 105 copies/ml recorded in our samples was also found to be higher compared to that of immunocompetent healthy subjects of Portugal,[16] Kuwait [17] and healthy blood donors of Switzerland.[18] However, the mean viral DNA load, if compared with observed mean values in three Italian immunocompetent subjects [19] and healthy adult women of USA,[20] was found to be lower.

We have also observed that the mean viral load in urine (1.07 × 106) was higher than that in the blood (7.77 × 105) samples of apparently healthy tribal individuals [Table 2]. The JCV is found to infect an individual in early childhood, after which it can remain in a latent state in the kidney [21] and is shed in the urine throughout the entire life without any noticeable clinical symptoms in the individual. Reactivation of JCV with the resulting urinary excretion of viruses readily occurs during conditions of immune suppression or immunocompromization. It has also been suggested that JCV could spread within normal brain tissue through blood or infected immune cells like B-lymphocytes.[22] Further studies are underway to confirm the effect of high viral load in both urine and peripheral blood leucocytes in the subsequent development of pathogenic conditions in CNS of the affected subjects.

Our study was aimed at quantitation of JCV load in body fluids of immunocompetent normal subjects. It has long been suggested that nutritional deficiency can lead to an immunodeficient condition in humans, which in turn enhances the susceptibility towards infection and disease condition.[23] Further studies are required to corroborate nutritional status of the studied tribal groups with that of the high viral load in the body fluids of the population studied.

In conclusion, the extent of the viral load in immunocompetent individuals was reported for the first time in tribal groups of this area suggesting a relatively higher amount of viral load present in the infected individuals. Given the ubiquitous nature and pathogenic potential of JCV in human subjects, further study can relate the observed viral loads in this region and their clinical implications.

~ Acknowledgements

The study was supported by a research grant from Council of Scientific and Industrial Research (CSIR), New Delhi, India awarded to the corresponding author (Sanction number 37 (1446)/10/EMR-II). Authors also acknowledge the help from Dr. Nripendranath Mandal, Molecular Medicine Division, Bose Institute, Kolkata, West Bengal in real-time PCR assays.